Identification of a Novel Two-Peptide Lantibiotic from Vagococcus fluvialis

ABSTRACT Infections caused by multiresistant pathogens have become a major problem in both human and veterinary medicine. Due to the declining efficacy of many antibiotics, new antimicrobials are needed. Promising alternatives or additions to antibiotics are bacteriocins, antimicrobial peptides of bacterial origin with activity against many pathogens, including antibiotic-resistant strains. From a sample of fermented maize, we isolated a Vagococcus fluvialis strain producing a bacteriocin with antimicrobial activity against multiresistant Enterococcus faecium. Whole-genome sequencing revealed the genes for a novel two-peptide lantibiotic. The production of the lantibiotic by the isolate was confirmed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry, which revealed distinct peaks at 4,009.4 m/z and 3,181.7 m/z in separate fractions from reversed-phase chromatography. The combination of the two peptides resulted in a 1,200-fold increase in potency, confirming the two-peptide nature of the bacteriocin, named vagococcin T. The bacteriocin was demonstrated to kill sensitive cells by the formation of pores in the cell membrane, and its inhibition spectrum covers most Gram-positive bacteria, including multiresistant pathogens. To our knowledge, this is the first bacteriocin characterized from Vagococcus. IMPORTANCE Enterococci are common commensals in the intestines of humans and animals, but in recent years, they have been identified as one of the major causes of hospital-acquired infections due to their ability to quickly acquire virulence and antibiotic resistance determinants. Many hospital isolates are multiresistant, thereby making current therapeutic options critically limited. Novel antimicrobials or alternative therapeutic approaches are needed to overcome this global problem. Bacteriocins, natural ribosomally synthesized peptides produced by bacteria to eliminate other bacterial species living in a competitive environment, provide such an alternative. In this work, we purified and characterized a novel two-peptide lantibiotic produced by Vagococcus fluvialis LMGT 4216 isolated from fermented maize. The novel lantibiotic showed a broad spectrum of inhibition of Gram-positive strains, including vancomycin-resistant Enterococcus faecium, demonstrating its therapeutic potential.

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Infections caused by multi-resistant pathogens have become a major problem in both human 12 and veterinary medicine. Due to the declining efficacy of many antibiotics, new 13 antimicrobials are needed. A promising alternative or addition to antibiotics are bacteriocins; 14 antimicrobial peptides of bacterial origin with activity against many pathogens including 15 antibiotic-resistant strains. From a sample of fermented maize, we isolated a Vagococcus 16 fluvialis strain producing a bacteriocin with antimicrobial activity against multi-resistant 17 Enterococcus faecium. Whole-genome sequencing revealed a novel two-peptide lantibiotic. 18 Production of the lantibiotic by the isolate was confirmed by matrix-assisted laser desorption 19 ionization-time of flight (MALDI-TOF) mass spectrometry, which revealed distinct peaks at 20 4009.4 m/z and 3181.7 m/z in separate fractions from reversed-phase chromatography. The 21 combination of the two peptides resulted in a 1200-fold increase in potency, confirming the 22 two-peptide nature of the bacteriocin, named vagococcin T. The bacteriocin was 23 demonstrated to kill sensitive cells by the formation of pores in the cell membrane, and its 24 inhibition spectrum covers most Gram-positive bacteria, including multi-resistant pathogens. 25 To our knowledge, this is the first bacteriocin characterized from Vagococcus. 26

Importance 27
Enterococci are common commensals in the intestines of humans and animals, but in recent 28 years, they have been identified as one of the major causes of hospital-acquired infections due 29 to their ability to quickly acquire virulence and antibiotic resistance determinants. Many 30 hospital isolates are multi-resistant thereby making current therapeutic options critically 31 limited. Novel antimicrobials or alternative therapeutic approaches are needed to overcome 32 this global problem. Bacteriocins, natural ribosomally synthesized peptides produced by 33 bacteria to eliminate other bacterial species living in a competitive environment, provide such 34 an alternative. In this work, we purified and characterized a novel two-peptide lantibiotic 35 produced by Vagococcus fluvialis isolated from fermented maize. The novel lantibiotic

Introduction 39
Enterococci such as E. faecium and E. faecalis are regular commensals of human and 40 animal intestines (1, 2). However, in recent years enterococci have become a concern in both 41 human and veterinary medicine as they have emerged as some of the most prevalent 42 nosocomial pathogens (3,4). In addition to their ability to effectively acquire, harbor, and 43 distribute antimicrobial resistance (AMR) determinants, enterococci are robust and able to 44 survive on non-biotic surfaces for prolonged periods (5, 6). There is increasing evidence that 45 overuse of antibiotics is a primary selection pressure for the acquisition and dissemination of 46 antibiotic resistance in bacteria (7). To reduce the dissemination of AMR and to combat 47 resistant bacteria, alternatives to antibiotics are needed. One such promising alternative is 48 bacteriocins -natural proteinaceous compounds produced by bacteria with antimicrobial 49 activity mostly against closely related species including pathogenic and antibiotic-resistant 50 strains. 51 Small bacteriocins (less than 10 kDa) are classified based on their biosynthesis: post-52 translationally modified bacteriocins belong to class I while unmodified bacteriocins are 53 members of class II (8,9). Lanthipeptides, which belong to class I, are characterized by 54 thioether linkages formed between cysteines and dehydrated serine and threonine residues to 55 yield lanthionine and methyllanthionine, respectively (10). The organization of the ring 56 structures then recognizes a specific target on sensitive cells such as lipid II, which is the 57 docking molecule for most lantibiotics (11). The bacteriocin producer must protect itself from 58 the lethal action of its own bacteriocin. For lantibiotics, self-immunity is achieved by the 59 production of immunity proteins commonly named LanI and/or LanFE(G) (12, 13). The 60 proteins LanFE(G) compose a specialized ABC-transporter that mediates the efflux of mature 61 lanthipeptides from the cell, while LanI is thought to protect the producer extracellularly 62 against the secreted lanthipeptide (12).

63
Lantibiotics are further subdivided into at least two types based on the differences in 64 modification enzymes (14). Type I lanthipeptides, of which nisin is the founding member, use 65 two separate enzymes for the dehydration (LanB) and cyclization (LanC) steps that produce 66 the (methyl)lanthionine rings. Type II employs a single bifunctional enzyme (LanM) that 67 catalyzes both steps (10,14). LanM modification enzymes usually carry out the modification 68 of two-peptide lantibiotics, which each consists of two different peptides exhibiting 69 considerable synergy when combined but having little or no activity when assessed 70 individually ( isolates originated from the wounds of animals (pigs, horses, cattle) and from human clinical 79 cases (20, 21). However, the species has also been isolated from the urine of healthy cattle 80 and described as a potential probiotic in fish (22,23). In this work, we describe the discovery 81 and characterization of a novel two-peptide lantibiotic produced by Vagococcus fluvialis. production. Repetitive element PCR (rep-PCR) was performed to examine the genetic 100 similarity of these isolates. Nine unique DNA band profiles were observed after gel 101 electrophoresis (data not shown). One representative from each group was selected for whole-102 genome sequencing to identify novel bacteriocin genes. The genomes were analyzed for 103 bacteriocins by BAGEL4 and antiSMASH (29,30). The analysis revealed that all but two 104 isolates had genes for previously characterized bacteriocins known to be active against 105 enterococci; subtilosin A (31), ericin S (32), enterolysin A (33), and NKR-5-3B (34). One of 106 the two isolates with a potentially novel bacteriocin was a strain isolated from fermented 107 maize, the genome of this isolate contained a gene cluster with an organization similar to two-108 peptide lantibiotic gene clusters. The best database hit for the predicted bacteriocin was the 109 lantibiotic flavecin from Ruminococcus flavefaciens (35) with only 45% identity, suggesting 110 that the isolate, identified as Vagococcus fluvialis, likely produced a novel two-peptide 111 lantibiotic. 112

Genome analysis and identification of the vagococcin T gene cluster 113
The search for putative bacteriocin genes by antiSMASH resulted in the identification 114 of a type II lantibiotic gene cluster (Fig. 1). Two bacteriocin genes vcnA1 and vcnA2 were 115 identified and predicted to represent the a (vcnA1) and b (vcnA2) peptides of a two-peptide 116 lantibiotic hereafter named vagococcin T. Located downstream of each of the vcnA1 and 117 vcnA2 genes are genes encoding lantibiotic biosynthesis proteins vcnM1 and vcnM2, 118 respectively. Both gene products, VcnM1 and VcnM2, showed sequence similarity with 119 MrsM, the modification enzyme for the lantibiotic mersacidin (36). The predicted function of 120 all proteins encoded in the vcn gene cluster is listed in Table 2. 121 (blue). Lantibiotic transporter gene with leader removal function (gray) is located downstream 125 of vcnM1 and upstream from vcnI, encoding a potential immunity protein (yellow). Other 126 genes involved in bacteriocin immunity are located at the beginning of the cluster. A group of 127 genes resembling a quorum-sensing system (red) is located at the end of the cluster. 128 The vcnT gene is located downstream of vcnM1 and encodes a C39 peptidase that 129 shows 45% identity with MrsT, the mersacidin transport enzyme which cleaves the leader 130 after the GG/GA motif -a typical cleavage site for many bacteriocin leaders (37). A GG-131 motif is indeed present in both VcnA1 and VcnA2 prepeptides ( Fig. 2A). The mature peptides 132 showed the highest homology to flavecin FlvA1a and FlvA2b peptides (42% and 46%, 133 respectively) (35). Sequence alignment of Vcn Ta with other lantibiotic α-peptides ( Fig. 2B) 134 showed that Vcn Ta contain the same CTxTxEC conserved motif believed to be essential for 135 lipid II docking (38). Similarly, the conserved sequence (CPTxxCt/sxxC) typical for all b-136 peptides was found in Vcn Tb (Fig. 2B). 137 with BoxShade; black and gray shading corresponds to identical and similar amino acids, 145 respectively. 146 The types of immunity genes present in lantibiotic gene clusters vary, and the encoded 147 immunity proteins often show little sequence identity with each other (39). Two genes of the 148 LanFE(G) immunity system are present in the vcn cluster, vcnF and vcnE, located at the start 149 of the operon. VcnF showed 47% identity with the ATP-binding domain NisF of the NisFEG 150 transporter, and contained the conserved sequences for both Walker A and B motifs (40). 151 The last four genes in the cluster resembled an analog to the Fsr quorum-sensing system 152 of E. faecalis; this type of quorum-sensing system has not previously been identified in other 153 lantibiotic clusters (41). The product of the first ORF, designated vcnR, showed 39% identity 154 to the response regulator (RR) FsrA (see Table 2). An FsrB homologue is encoded by the 155 gene designated vcnQ2 with 36% identity (Q for quorum). The third component, a sensor 156 histidine protein kinase (HPK) encoded by vcnK showed 35% identity with FsrC. Search for 157 small reading frames that could encode the pheromone component of the quorum-sensing 158 system revealed a small ORF between vcnQ2 and vcnK. The product of this ORF gave no hits 159 to any known peptides by BLAST search, however, sequence alignment showed 37% identity 160 with FsrD, the gelatinase biosynthesis-activating pheromone (GBAP) prepeptide (42). It is 161 therefore possible that the processed product of vcnQ1 is a pheromone. 162 Another small ORF located between vcnT and vcnR also showed no sequence 163 homology to known proteins by BLAST-search, however, the gene product had similar size, 164 charge, and hydrophobicity as known lantibiotic immunity proteins. LanI proteins of 165 comparable physicochemical properties include EciI, PepI, and LasJ, the LanI immunity 166 proteins for epicidin 280, pep5, and lactocin S (39). The ORF located between vcnT and vcnR 167 was therefore named vcnI and is further discussed in the Discussion section below. 168 Because of the novelty of vagococcin T, the antimicrobial produced by the isolate of V. 171 fluvialis was chosen for further characterization. Cell-free supernatants from the isolate 172 contained an antimicrobial substance which was heat-stable and sensitive to proteinase K 173 (data not shown), properties expected for bacteriocins, like vagococcin T (43). 174

Purification of bacteriocin 175
Purification of the predicted two-peptide lantibiotic produced by V. fluvialis was 176 achieved with a three-step purification scheme consisting of ammonium sulfate precipitation, 177 cation-exchange chromatography and reverse-phase chromatography (RPC) (44). During the 178 RPC elution, two peaks corresponding to 29% and 36% of isopropanol were observed in the 179 elution profile (Fig. 3). Collected fractions were assayed against E. faecium LMG 20705; low 180 antimicrobial activity of 400 BU/ml was found only in the second peak (fractions 26 to 30), 181 which would be expected due to the separation of the two peptides into separate fractions 182 (45). To test this notion, fractions 21 to 24 were individually combined with fractions 26 to 30 183 in 1:1 (v/v) ratio to find any combination of fractions exhibiting synergy (Fig. S1). Indeed, the 184 highest synergy was observed between fractions 23 and 28 which in combination had 185 antimicrobial activity of 51 200 BU/ml, representing a 1200-fold increase in activity with 186 yield of 128% (Table 2)  With purified bacteriocin, the biological activity of vagococcin T against number of 196 bacteria was determined using the spot-on-lawn assay (Table 3). Lantibiotics are known to be 197 very potent against Gram-positive bacteria but with limited activity against Gram-negative 198 bacteria as observed for nisin (46), lichenicidin (47)

Molecular Mass and Bacteriocin Identification 208
Given the synergism of fraction 23 on fraction 28, these fractions were analyzed further 209 using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass 210 spectrometry. The acquired spectra revealed the presence of only one distinct peak in each 211 fraction. A peak at 4009.5 m/z can be seen in fraction 23 (Fig. 4A), which correlated well 212 with the mass predicted for one of the two peptides by antiSMASH (30) (assuming 1 213 unmodified Ser/Thr). The peak in fraction 28 (Fig. 4B)  is shown in Figure 5. These results confirm that the antimicrobial activity produced by V. 218 fluvialis was indeed caused by the predicted two-peptide lantibiotic vagococcin T. 219 Vcn Ta and Vcn Tb were deduced from the known structures of other two-peptide lantibiotics.

Pore-forming nature of vagococcin T 230
To assess whether vagococcin T is a pore former, propidium iodide (PI) assay was 231 conducted. PI is a membrane impermeant dye which increases its fluorescence 232 efficiency/quantum yield when bound to double-stranded DNA (49). After exposing the 233 indicator strain to the known pore-forming lantibiotics nisin A and nisin Z in the presence of 234 extracellular PI, an increase in emission was detected (Fig. 6). Similar results were also 235 obtained for vagococcin T, implying that vagococcin T has a similar mode of action involving 236 pore-formation. The negative control micrococcin P1, a bacteriocin which kills cells by 237 inhibition of protein synthesis (50) the pore-forming nisin A and nisin Z. Micrococcin P1, a non-pore-forming bacteriocin, was 244 used as a negative control. 245 To further corroborate our results that vagococcin T is membrane-active, the indicator 246 cells exposed to vagococcin T were examined by scanning electron microscopy (SEM). Clear 247 differences were observed for bacteriocin-treated compared to untreated cells (Fig. 7). Treated 248 cells appeared collapsed/shriveled, suggesting loss of turgor pressure. Irregular dark spots 249 were visible on some cells, possibly indicating pores or damage to the cell envelope. In indicated by red arrows. 259

Stress response involved in resistance to vagococcin T 260
Resistant colonies of E. faecium LMG 20705 were occasionally visible within the 261 inhibition zones of vagococcin T. The increased tolerance to vagococcin T of four randomly 262 selected spontaneous mutants was tested and showed a 64-to 256-fold increase in MIC 263 (minimum inhibitory concentration) compared to the wild-type (Table 4). The frequency of 264 resistant mutants was estimated to be 8.7 x 10 -7 based on plating techniques. Whole-genome 265 sequencing was performed on the four mutants to identify the possible mechanism for the 266 increased tolerance to vagococcin T. Three of the four mutants had mutations in liaF (M1-267 M3), two with non-conservative missense mutations (Ile108Asn, Trp141Ser) and one with a 268 frameshift from amino acid position 9 (Val9fs, M2). Several mutations were found in various 269 genes of mutant M4, none of which could be directly linked to the increased tolerance to 270 vagococcin T, see Table 4. liaF encodes a negative regulator of LiaRS, a two-component 271 regulatory system involved in cell envelope stress response induced by lipid II-interacting 272 antimicrobials (51). We examined the cross-resistance of liaF mutants to other membrane-273 active bacteriocins -nisin A and garvicin KS (44). As expected, both nisin A and garvicin KS 274 showed reduced bioactivity (4-to 32-fold) toward the mutants compared to the wild-type 275 strain. 276 Bacteriocins are a promising alternative to traditional antibiotics, as they display 282 activity against antibiotic-resistant pathogens and have many desirable properties for the 283 control of microorganisms. They are often produced by probiotic species with GRAS 284 (generally regarded as safe) status, have high potency, and low toxicity (52). In addition, 285 bacteriocins are arguably more easily amenable to biotechnological manipulation as they are 286 defined by structural genes. Given the high potency and potential clinical applications of 287 bacteriocins, we sought to find new bacteriocins with possible therapeutic use. To this end, 288 we screened for bacteriocin producers in fermented fruits and vegetables that inhibited the 289 growth of the indicator strain, a multi-drug resistant E. faecium. From a sample of fermented 290 maize, we successfully isolated a strain of V. fluvialis producing a two-peptide lantibiotic 291 named vagococcin T. 292 To our knowledge, vagococcin T was first bacteriocin identified from the genus 293 Vagococcus. The two bacteriocin genes, vcnA1 and vcnA2, are separated by a vcnM gene, 294 which is an unusual arrangement -two-peptide bacteriocin genes are most often located 295 adjacent to each other in tandem. Because of the low sequence similarity of the two 296 vagococcin T prepeptides (20% sequence identity), each of the two vcnM gene products are 297 likely dedicated to modifying its cognate bacteriocin peptide. Upstream of the bacteriocin 298 genes in the same operon is the gene pair vcnFE encoding an ABC-transporter which likely 299 has a dual role, in export of the bacteriocins peptides and immunity; a property which is 300 common for other lantibiotics including nisin, mersacidin and lacticin 3147 (39). At the end 301 of the vcn cluster is an operon encoding proteins with homology to the Fsr quorum-sensing 302 system from E. faecalis. In the Fsr system, the FsrD propeptide is exported and processed by 303 FsrB into a small 11 amino acid cyclic peptide pheromone. A membrane-bound sensor HPK 304 FsrC (VcnK) then responds to the pheromone and activates the intracellular RR FsrA (VcnR) 305 (30). VcnQ2 and VcnQ1 show 35% and 37% sequence identity with FsrB and FsrD, 306 respectively (Table 1). The majority of circular peptide pheromones have been reported to 307 form a thiolactone linkage between the C-terminal amino acid (methionine, phenylalanine, or 308 leucine) and a cysteine located three or four residues from the N-terminal cleavage site (53). 309 However, the peptide processed from FsrD contains a lactone linkage between the C-terminal 310 methionine and the hydroxyl group of a serine residue (42). In addition, an autoinducing 311 peptide containing a lactone ring between the C-terminal phenylalanine and a serine residue 312 has been identified in S. intermedius (54). VcnQ1 may be processed similarly, forming a 313 lactone linkage between serine and the C-terminal phenylalanine. Interestingly, the closest 314 homologue to VcnQ1 was found to be an unannotated orf (159 nt) in the locus of the circular 315 bacteriocin enterocin NKR-5-3B (Ent53B) produced by the strain E. faecium NKR-5-3 316 (GenBank Accession: LC068607) (55). The orf is arranged similarly to vcnQ1 between genes 317 encoding an HPK and a FsrB-like protein (orf5 and orf6). The predicted mature product of 318 this orf contains an 11 amino acid sequence showing 73% identity (100% similarity) to the 319 putative VcnQ1-derived pheromone. E. faecium NKR-5-3 produces multiple bacteriocins; 320 enterocins NKR-5-3A, B, C, D, and Z (Ent53A, Ent53B, Ent53C, Ent53D, and Ent53Z) (56).

321
An inducing peptide Ent53D has been shown to regulate the transcription of the 322 aforementioned bacteriocins except for NKR-5-3B (56). A derivative of the unannotated orf 323 in E. faecium NKR-5-3 genome may be involved in the regulation of NKR-5-3B. However, it 324 is presently not known if VcnQRK constitutes a functional quorum-sensing system in V. 325 fluvialis; characterization of the vcn regulatory system is beyond the scope of the present 326 study.

327
The production of vagococcin T by the V. fluvialis isolate was confirmed by 328 bacteriocin purification and MALDI-TOF MS. Vagococcin T was purified from the cell-free 329 supernatant using a common purification scheme for bacteriocins involving ammonium 330 sulfate precipitation followed by cation-exchange-and reversed-phase chromatography. The 331 elution profile from reversed-phase chromatography showed two distinct peaks, indicating the 332 presence of a two-peptide bacteriocin. Indeed, when assayed individually, only fraction 28 333 exhibited some antimicrobial activity (400 BU/ml) against the indicator strain. However, 334 when all combinations of fractions were assayed (fractions 20 to 30), a significant increase in 335 potency (51 200 BU/ml) was observed for the combination of fractions 23 and 28. Despite not 336 corresponding to the two peaks in the elution profile, the high synergy observed for the 337 combination was strong evidence of a two-peptide bacteriocin. 338 Mass determination of each fraction revealed a single distinct peak at 4009.4 m/z and 339 3181.69 m/z for fractions 23 and 28, respectively. Analysis of V. fluvialis genome by the 340 RiPP mining tool antiSMASH (57) identified a lanthipeptide gene cluster encoding two 341 putative lanthipeptide precursors. In addition to predicting lanthipeptide genes, antiSMASH 342 predicts the leader cleavage site, dehydrations, crosslinks, and the expected masses. The mass 343 predicted for Vcn T⍺ (4010.6 Da), assuming one unmodified serine or threonine, 344 corresponded well with the measured value of 4009.4 m/z. However, the mass predicted for 345 Vcn Tβ (3111.6 Da) was approximately 71 Da lower than the mass obtained by MALDI-TOF 346 MS. The reason for this discrepancy is likely caused by inaccurate leader peptide prediction. 347 The predicted Vcn T⍺ leader peptide is a typical double-glycine-type leader with a GG| 348 cleavage site, while the Vcn Tβ leader cleavage site was predicted to be (G)GA|. The 349 predicted mass of Vcn Tβ with the addition of alanine is 3181.5 Da which is consistent with 350 the measured mass of 3181.67 m/z. The close correspondence between the measured and the 351 theoretical masses provides strong evidence that the purified bacteriocin vagococcin T is the 352 gene product of vcnA1 and vcnA2. Predicted structures of Vcn Ta and Vcn Tb peptides are  353 consistent with the structures of other two-peptide lantibiotics (Fig. 5).

354
The ⍺-peptide of most two-component lantibiotics employs lipid II as a docking 355 molecule to exert its antimicrobial activity (58, 59). A lipid II-binding motif was found in Vcn 356 T⍺ (see Fig. 2B), suggesting a lipid II-dependent mode of action of vagococcin T. It is 357 believed that the b-peptide of lipid II-targeting two-component lantibiotics binds to the 358 complex formed between lipid II and the ⍺-peptide, which then leads to pore formation. The 359 predicted mode of action involving pore formation was consistent with SEM showing E. 360 faecium with a shriveled appearance, lysed cells, and cell debris following the exposure to 361 vagococcin T (see Fig. 7). The extracellular matrix-like material is likely consisting of cell 362 debris cross-linked by the fixing agent. The pore formation property is further supported by 363 the fact that Vcn T showed a comparable pore-forming ability to nisin A, a known pore-364 forming lantibiotic (60, 61).

365
For many lantibiotics, the type of immunity system appears to correlate with the mode 366 of action of the lantibiotic (12, 13). It is believed that producers of pore-forming lantibiotics 367 require both the LanI and LanFE(G) components for immunity (13, 62). However, no LanI 368 component was immediately apparent in the vcn cluster, despite the evident pore-forming 369 mode of action of vagococcin T (see Fig. 6). On further analysis, a small ORF was found 370 downstream of vcnT, encoding a predicted transmembrane, cationic, 50 amino acid protein 371 (charge 5 at pH 7). The protein sequence shows no homology to known proteins but shares 372 similar properties with PepI, EciI, and LasJ (LanI component of Pep5, epicidin, and lactocin 373 S, respectively), all predicted transmembrane proteins, 57-69 amino acids in length with a 374 charge of 4-6 (at pH 7). Due to this similarity, we believe this ORF to be involved in 375 lantibiotic immunity and is thus named vcnI. 376 Upon challenging the E. faecium indicator strain to the bacteriocin we observed 377 resistant cells with a frequency of 8.7 x 10 -7 . Three randomly selected isolates with the 378 highest tolerance to vagococcin T all had mutations in liaF, a negative regulator (repressor) of 379 the LiaRS cell envelope stress response system (lipid II-interacting antibiotics response 380 regulator and sensor). Previous studies have shown that membrane-active antimicrobials 381 decouple the repression by LiaF, allowing the HPK LiaS and its cognate RR LiaR to trigger 382 genes involved in resistance (63). The effect of genetic disruption of liaF is likely similar to 383 the decoupling of LiaF-mediated repression. Orthologs of the Lia system exist in most 384 Firmicutes, and all systems investigated so far regulate the expression of genes that protect 385 the cell against perturbations in the cell envelope (51). In Bacillus subtilis, the LiaFSR system 386 is one of the primary response systems against lipid II-interacting antibiotics such as 387 vancomycin and bacitracin (64) but is also induced by cationic antimicrobial peptides, organic 388 solvents, and detergents (65-67). The genes regulated by the Lia system vary between 389 species; in Staphylococcus aureus the LiaRS homolog (VraSR) upregulates genes encoding 390 penicillin-binding proteins and proteins involved in teichoic acid synthesis, chaperones, and 391 membrane lipid biosynthesis, that together confer resistance to beta-lactam antibiotics (68-392 71). Even though the LiaFSR regulon in enterococci remains unknown, the LiaFSR system 393 has been implicated in resistance to daptomycin and antimicrobial peptides due to the 394 redistribution of cardiolipin microdomains away from the division septum (72, 73). All liaF 395 mutants displayed low-level cross-resistance to nisin A, another lipid II-interacting lantibiotic 396 (Table 4). These results confirm the role of LiaFSR in mediating resistance to vagococcin T 397 which further supports the lipid II-mediated mode of action of the bacteriocin. 398 The appearance of vagococcin T-resistant colonies of E. faecium exemplifies the 399 hardiness of enterococcal populations. Combination therapies will likely be needed to 400 effectively control enterococcal populations in the future. Formulations combining 401 bacteriocins with different modes of action have been developed and showed increased 402 potency and broader inhibition spectrum with a very low frequency of resistance (74-76).

403
In summary, in this work, we describe the isolation and characterization of a new two-404 component lantibiotic vagococcin T showing a broad antimicrobial spectrum against Gram-405 positive species, including multidrug-resistant strains. Furthermore, we show that mutations 406 in the liaF gene confer resistance to vagococcin T and other antimicrobials. This connection 407 highlights LiaF and the stress response system as an appealing target for future drug 408 development and combination therapies. Further work is required to establish the potential of 409 vagococcin T as a therapeutic in human or veterinary medicine. 410

Bacteriocin purification 446
The bacteriocin-producing strain was cultivated in 1 liter of BHI broth at 30°C for 24 447 hours. Cells were removed by centrifugation (10,000 g, 30 min, 4°C) and the bacteriocin was by centrifugation. Microcccin P1 was purified as previously described (77). 470 Spot-on-lawn assay was used to obtain the inhibition spectrum of purified vagococcin 471 T. Vagococcin T solution was prepared by mixing fractions with the highest synergy in a 1:1 472 ratio. Fresh overnight cultures were diluted 1:100 in 5 ml of BHI soft-agar and poured onto a 473 BHI agar plate. Once the layer solidified, 2 µl of vagococcin T solution was spotted on the 474 lawn. The plates were incubated overnight at 30°C and the inhibition zones were measured. 475

Propidium iodide assay 476
The pore-forming mode of action of vagococcin T was investigated using the propidium 477 iodide (PI) method. An overnight culture of the indicator was washed twice in phosphate-478 buffered saline (PBS) and adjusted to an OD600 of 0.7 with PBS in the wells of a black 479 microtiter plate containing 20 µM PI (final concentration) and vagococcin T. Fluorescence 480 was measured at 5-min intervals for 2 hours using a FLUOstar OPTIMA reader (BMG 481 LABTECH, Ortenberg, Germany) with excitation at 535 nm and emission at 617 nm. 482 483 MALDI-TOF mass spectrometry 484 485 MALDI-TOF MS was performed on an ultrafleXtreme mass spectrometer (Bruker 486 Daltonics, Bremen, Germany) operated in reflectron mode. The instrument was externally 487 calibrated with peptide calibration standard II (Bruker Daltonics, Bremen, Germany) and 488 positively charged ions in the range of 1000 to 6000 m/z were analyzed. RPC purified 489 fractions and matrix [a-cyano-4-hydroxycinnamic acid (HCCA)] were mixed in 1:1 ratio and 490 applied on a Bruker MTP 384 steel target plate (Bruker Daltonics, Bremen, Germany) for 491 analysis. 492

Scanning electron microscopy 493
The indicator strain was grown to mid-log phase (OD600 ~ 0.6) and incubated with 494 vagococcin T (10x MIC) for 2 hours at 37°C with gentle shaking. A culture with no 495 bacteriocin added was used a control. After incubation, cells were harvested by centrifugation 496 (10,000g, 5 min), washed twice in PBS and resuspended in fixing solution (1.25% w/v 497 glutaraldehyde, 2% w/v formaldehyde, PBS) for overnight incubation at 4°C. Fixed cells were 498 then washed three times in PBS and allowed to sediment/attach onto poly-L-lysine coated 499 glass coverslips at 4°C for 1 hour. Subsequently, attached cells were dehydrated with an 500 increasing ethanol series (30, 50, 70, 90, 96% v/v) for 10 min each and finally washed four 501 times in 100% ethanol. Cells were dried by critical-point drying using a CPD 030 critical 502 point dryer (BAL-TEC, Los Angeles, CA, USA). Coverslips were sputter coated with 503 palladium-gold using a Polaron Range sputter coater (Quorum Technologies, Lewes, UK).

504
Microscopy was performed on an EVO50 EP scanning electron microscope (Zeiss, 505 Oberkochen, Germany) at 20 kV and a probe current of 15 pA. 506

Mutant analysis 507
To characterize mutants of E. faecium LMG 20705 resistant to vagococcin T, a total of 508 20 plates were made as described for the spot-on-lawn assay. However, to avoid sequencing 509 clones of the same mutant, the lawn on each plate was prepared from genetically independent 510 cultures (inoculated with different single colonies). Colonies that were observed at or near the 511 center of the inhibition zone from vagococcin T following overnight incubation were picked. 512 Colonies from several agar plates were re-streaked to obtain pure cultures. The 513 resistance to vagococcin T was confirmed and quantified by determining the bacteriocin 514 activity towards the mutants compared to the wild type strain. Genomic DNA of mutant 515 strains was isolated with GenElute TM Bacterial Genomic DNA kit (Sigma-Aldrich, Saint-516 Louis, MO, USA) according to manufacturer's instructions and sent to Novogene (Novogene 517 Bioinformatics Technology Co., Ltd, Beijing, China) for sequencing (NovaSeq 150 bp paired-518 end). Reads from the wild type was assembled using SPAdes v3.15.3 to obtain reference 519 contigs. Snippy was used to identify variants by mapping the reads from mutant isolates to the 520 reference contigs using the default settings (82). 521

Accession number 522
The DNA sequence of vagococcin T gene cluster was submitted to GenBank under the 523 accession number OM959625. 524